University of Groningen
Influences of Complex Topography and Biochemistry on Mesenchymal Stem Cell
Differentiation
Yang, Liangliang
DOI:10.33612/diss.146104615
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Publication date: 2020
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Yang, L. (2020). Influences of Complex Topography and Biochemistry on Mesenchymal Stem Cell Differentiation. University of Groningen. https://doi.org/10.33612/diss.146104615
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107
CHAPTER 7
Synergistic Effect of Cell-derived
Extracellular Matrix and Topography on
Osteogenesis of Mesenchymal Stem
Cells
This chapter has been published in:
108 Abstract
Cell-derived matrices (CDM) are an interesting alternative to the conventional sources of extracellular matix (ECM) as CDM mimics better the natural ECM composition and are therefore attractive as a scaffolding material to be used for regulating the functions of stem cells. Previous research on stem cell differentiation has demonstrated that both surface topography and CDM have a significant influence. However, not much focus has been placed on elucidating possible synergistic effects of CDM and topography on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this study, Polydimethylsiloxane (PDMS)-based anisotropic topographies (wrinkles) with various topography dimensions were prepared and subsequently combined with native ECM produced by human fibroblasts that remained onto the surface topography after decellularization. The synergistic effect of CDM combined with topography on osteogenic differentiation of hBM-MSCs was investigated. The results showed that substrates with specific topography dimensions, coated with aligned CDM, dramatically enhanced the capacity of osteogenesis as investigated using immunofluorescent staining for identifying osteopontin (OPN) and mineralization. Furthermore, the hBM-MSCs on the substrates decorated with CDM exhibited higher percentage of YAP inside the nucleus, stronger cell contractility, and more formation of focal adhesion, illustrating that enhanced osteogenesis is partly mediated by cellular tension and mechanotransduction following the YAP pathway. Taken together, our findings highlight the importance of ECM mediating the osteogenic differentiation of stem cells, and the combination of CDM and topography will be a powerful approach for material-driven osteogenesis.
Keywords: extracellular matrix, topography, mesenchymal stem cells, osteogenic differentiation, mechanotransduction
109 7.1 Introduction
In vivo, cells directly interact with their surrounding microenvironment, the extracellular matrix, that is secreted by cells and composed of a complex mixture of polysaccharides and proteins. The ECM provides mechanical support and further introduces many biochemical and biophysical stimuli, including adhesion receptors, topographical cues, mechanical input for regulating cell response, such as cell adhesion/spreading, proliferation, migration, differentiation, or apoptosis1. Therefore, mimicking the interactions of the natural ECM and incorporate such approaches in the development of biomaterials enables studying of cells in a realistic and adaptable cell microenvironment in vitro2,3.
For topography, considerable research has highlighted the essential role of substrate topography on the cell behavior and lineage commitment of different types of stem cells 4,5, and this can be adjusted by the types and parameters of the topographical structures6–8. Furthermore, in vivo, there are many tissues composed of well-aligned and anisotropic architectures with nano- and microscale features (e.g., tendon, bone, nerve), therefore, it is important to mimic the anisotropic structure of bone ECM for biomaterials to study the influence on cell behaviors. Cells could sense surface parameters varying from 10 nm to 100 μm by contact guidance9,10. Previously, our group fabricated topography-containing surfaces and gradients with nano- and microscale and found that topography parameters have a significant effect on cells, including cell adhesion, elongation, orientation, migration, as well as differentiation of stem cells11–17. The aligned topographies were envisioned to represent better the ECM fiber morphology, which is important as the fibrous structure of the cellular microenvironment in vivo is essential for directing numerous cell functions18. In recent years, cellular adhesion protein/peptide have been applied as biomimicking matrices for improving the biocompatibility of biomaterials by chemical attachment or physisorption, e.g., ECM component (collagen19, fibronectin20), Matrigel21, GRGDS22, GYIGSR23, IKVAV24, N-cadherin25, and E-cadherin26. However, individual ECM protein/peptide cannot completely mimic the complexity of the endogenous ECM and often lack the powerful topographical stimulus. Therefore, diverse ECM proteins that mimic the composition of ECM in vivo should be investigated to gain insights in how to further push the control of stem cell differentiation.
In recent years, an increasing interest emerged for developing CDM, derived from native tissues or cells cultured in vitro, to form a microenvironment that mimics a native niche27,28. The CDM represents best the cellular microenvironment in tissues29. The decellularized ECM is the non-cellular component containing various cell secreted macromolecules that provides a natural scaffold of similar biological and structural make-up. While the CDM may differ significantly depending on the origin (cell type and tissue), it mostly consists of proteoglycans, such as growth factors, glycosaminoglycans (GAGs), and matrix proteins, e.g., collagen (Col I), fibronectin (Fn), elastin, vitronectin, and laminin30. Previously it was demonstrated that bio-chemical and biophysical cues can be conserved after the removal of the cellular components while removing components such as DNA and cellular components that trigger immune responses31, which makes the decellularized ECM a (stem) cell substrate that is close to the natural environment27,32,33. CDMs could be generated from different cell sources, e.g., fibroblasts34,35, mesenchymal stem cells36,37, and pluripotent stem cells33. Compared to other cell types, human dermal fibroblasts have several advantages, for example, readily isolated, substantial secretion of ECM biomolecules38, and the matrices generated from fibroblasts are much stronger than collagen or fibrin gels, which are often the reconstituted ECM components of choice39. Leach et al.32 found that cell-derived extracellular matrices from bone marrow-derived mesenchymal stem cells, human dermal fibroblasts, and adipose stromal cells, all promote the osteogenic differentiation, therefore, human dermal fibroblasts is a highly interesting cell source that secrets substantial amounts of relevant ECM.
Much work so far has focused on the influence of CDM derived from cells on multi-lineage differentiation potential, for instance, Tuan and co-workers40 showed that CDM from mesenchymal stem cells (MSCs) dramatically enhanced attachment, proliferation, migration, and differentiation of MSCs (osteogenic and adipogenic differentiation), as compared to surfaces coated with Col I. Furthermore, Li et al.33 found that CDM from aggregates of pluripotent stem cells modulate neurogenesis through biological cues and biophysical properties. These studies indicate that CDM scaffolds are interesting for tissue engineering with many possibilities in applications for tissue engineering and regenerative medicine. However, very few studies have been undertaken for investigating potential synergistic effects of topography and CDM on stem cell osteogenesis.
In this study we aim to explore the synergism of anisotropic topography and fibroblast-derived CDM on the osteogenesis of hBM-MSCs. We hypothesized that the combination of topography and
fibroblast-110
derived ECM would significantly enhance the fate commitment toward osteogenesis of hBM-MSCs. For this purpose, PDMS-based anisotropic topographies varying in wavelengths and amplitudes were used, which were found to interact with hBM-MSCs in our previous work14. As depicted in Figure 1, MSCs were seeded on topography substrates decorated with the fibroblast-derived ECM after decellularization, and allowed to expand following the standard culture procedure. The endogenies ECM proteins, Col I and Fn were confirmed to be present by using immunofluorescence labeling. The degree of osteogenic differentiation of hBM-MSCs was analyzed using immunofluorescently labeling of OPN and mineralization by staining the mineral phase with Alizarin red. Furthermore, the formation of focal adhesion and cell contractility, activation of YAP signal pathway were analyzed in depth to reveal the mechanism of MSC response to the wrinkle substrates decorated with CDM. We found that substrates decorated with CDM had a remarkable effect on cell orientation and cell area, and that there is a synergistic effect of specific topography combined with CDM on the osteogenic differentiation of hBM-MSCs, probably mediated by the focal adhesion, cytoskeletal contractility, and YAP signaling pathway.
Figure 1. Schematic representation of the preparation process of the CDM. Fibroblast-derived extracellular matrix was obtained through a decellularization process of cultured fibroblasts. Then onto the matrix hBM-MSCs were seeded to investigate to co-effect of topography and CDM on osteogenesis. 7.2 Methods
7.2.1 PDMS substrate preparation
PDMS substrates were prepared as described previously14. Briefly, PDMS was prepared by combining a elastomer prepolymer and cross-linking agent (Sylgard 184, Dow Corning) in a ratio 10:1 by weight and the mixture was degassed to remove air for 15 min. The PDMS was subsequently further cured at 70 °C overnight.
7.2.2 Preparation of PDMS aligned topography substrates (molds)
PDMS substrates with aligned wrinkle topography were prepared similar as previously reported14. PDMS elastomeric substrate was uniaxially stretched to 120−130% of the initial length and subsequently oxidized by air plasma (Plasma Activate Flecto 10 USB, maximum intensity) using different pressures and variations in time depending on the desired features. Afterwards, the strain was released and anisotropic wrinkle with
111 varied wavelength and amplitude was formed. Table. 1 summarizes the conditions for wrinkle substrate preparations. The samples prepared in this step have been used as the molds for imprinting.
Table 1. Conditions for wrinkle preparation on PDMS substrates used for molds
W and A reflect wavelength and amplitude, respectively, and are depicted in μm. The substrates were further indicated as W0.5, W3, and W10.
7.2.3 Imprinting
To exclude chemical and mechanical variations originating from the different preparation procedures, a mixture of prepolymer and cross-linking agent (ratio of 10:1, weight) was poured onto the surface of the wrinkle substrates acting as the mold, prepared in last step, and was cured at 70 °C for overnight. Afterwards, the freshly prepared substrates were detached from the mold, and treated with air plasma before cell seeding at 500 mTorr for 1 min. The Flat control sample was treated similar as the imprints indicated above (10:1 for prepolymer and cross-linking agent, same curing and oxidization process), to guarantee that the substrates keep similar surface physicochemical properties.
7.2.4 Topography Characterization
Topography was characterized by atomic force microscope (AFM, Nanoscope V Dimension 3100 microscope, Veeco, United States) using tapping mode approach in air (DNP-10 tip). The features were analyzed by NanoScope Analysis software.
7.2.5 Fibroblast-derived extracellular matrix formation
Substrates bearing decellularized ECM were prepared similarly as previously reported with modifications38,41. Briefly, human dermal fibroblasts were seeded at the density of 2 × 104 cells/well in 24-well plate containing the different PDMS substrates (Flat and topography), and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Gibco), 0.1% ascorbic acid 2-phosphate (Sigma), and 1% glutamax (Gibco). Every three days the medium was refreshed. A confluent cell layer reached after 10 days and was washed with phosphate-buffered saline (PBS) twice and subsequently decellularized by incubation with a 0.5% Triton X-100 solution and 20 mM NH4OH in PBS at 37 °C for 10 min. Samples were afterwards treated with a 10 μg/ml solution of DNase I (Roche) at 37 °C for 2 h to get rid of any DNA contamination. The decellularized CDM was gently washed with PBS five times to completely remove all of the sacrificial fibroblasts, and the resulting CDM was immediately used or kept under sterile conditions at 4 °C before use.
7.2.6 Cell Culture
hBM-MSCs from Lonza (passage 2) were cultured in growth medium supplemented with Alpha modified Eagle medium (Gibco), 10% fetal bovine serum (Gibco), 0.1% ascorbic acid 2-phosphate (Sigma), and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37 °C with 5% CO2. Every three days the culture medium was refreshed and cells were passaged or harvested at approximately 80% confluence. The confluent cells were subcultured by trypsinization. hBM-MSCs of passage 4 were used for the next experiments.
Substrate prepolymer and Ratio of curing agent Stretched percentage (%) Operating pressure (Torr) Plasma time (s) W0.5/A0.05 10:1 30 14 60 W3/A0.7 10:1 30 0.025 20 W10/A3.5 10:1 20 0.025 650
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7.2.7 Immunostaining
PDMS substrates were washed with 70% ethanol for sterilization and put in 24-well plates. The substrates were washed with PBS prior to use. Afterward, hBM-MSCs were seeded at a density of 1 × 104 cells/well. For immunostaining, hBM-MSCs were rinsed with Dulbecco's PBS (DPBS), and subsequently fixed using 3.7% paraformaldehyde (PFA) solution in PBS for 20 min. Afterward, the membrane of the cell was permeabilized using a 0.5% Triton X-100 solution for 3 min and blocked with 5% bovine serum albumin (BSA) in PBS solution for 30 min. The cells were subsequently incubated with a primary antibody for collagen (Sigma, 1:100), fibronectin (Sigma, 1:100), OPN (Developmental Hybridoma Bank, MPIIIB10, 1:100), vinculin (clone hVin-1, Sigma, 1:100), phosphorylated myosin light chain (pMLC, Cell Signaling, 3675, 1:200), YAP (Santa Cruz Biotechnology, SC-101199, 1:100) for 1 h, followed by treatment with secondary antibody Rhodamine RedTM-X-labeled goat-anti-mouse antibody (Jackson Immunolab, 1:100). Finally, the nucleus and cytoskeleton were stained with DAPI and FITC/TRITC-phalloidin, respectively, by incubation for another 1 hour. For imaging of the cells the TissueFaxs (TissueGnostics GmbH, Vienna, Austria) was used. Vinculin, pMLC, and YAP staining were imaged with a LEICA TCS SP2 confocal laser scanning microscopy (CLSM) equipped with a 40× NA 0.80 water immersion objective. Additionally, focal adhesion determinations were performed by analysis of the images using an online Focal Adhesion Analysis Server42, and elongation of Focal Adhesion was said to be the ratio between the length on the major axis to the width of the minor axis, thereby cell with a perfect circle shape has an elongation of 1 (also applied for cell elongation). The myosin fluorescent intensity was determined as previously reported.43
7.2.8 Osteogenic Differentiation of hBM-MSCs
hBM-MSCs were cultured on the different samples at a cell density of 1×104 cells/well in 24-well plate. Cells were incubated at 37 °C, 5% CO2 and after 24 h the growth medium was exchanged for osteogenic induction medium (OM), which was composed of growth medium supplemented with 10 mM glycerophosphate (Sigma) and 100 nM dexamethasone (Sigma). The cells were cultured over a period of 14−21 days and replacement of the medium was done every three days.
7.2.9 Mineralization identification by Alizarin Red Staining
The mineralization of the ECM was analyzed by Alizarin Red staining after culturing the cells for 21 days under differentiation conditions. The samples were washed with PBS twice, fixed with 4% PFA for 15 minutes and incubated with 0.1% Alizarin Red solution at room temperature for 30 minutes. Cells were two times washed with PBS before imaging. For quantification of the mineralization, the stained nodules were extracted for 30 minutes with 10% cetylpyridinum chloride in 10 mM sodium phosphate buffer at room temperature. The absorbance was determined by a microplate reader (BMG LABTECH, Offenburg, Germany) at 540 nm to determined quantitatively the amount of stain present. Normalization was performed for the results by accounting for the cell number in each well. The number of cells was calculated by quantitative analysis of DAPI positive nuclei using TissueQuest software after imaging with TissueFaxs-Tissue-Gnostics microscopy setup in a high-throughput manner.
7.2.10 Statistics
Data are given as mean values ± standard deviation (SD). Origin 9.0 software was used for statistical analysis. One way analysis of variance (ANOVA) with Tukey’s test was used for all data to determine differences between groups. *P < 0.05, **P < 0.01, and ***P < 0.001.
7.3 Results
7.3.1 Topography-CDM substrate fabrication and characterization
To determine the synergism between topography and CDM on the differentiation behavior of stem cells, CDM was prepared by cultivating fibroblasts on the substrates with different aligned topographies for 10 days, which were subsequently decellularized using a chemical approach.
In this study, PDMS substrates with aligned topographies were prepared as described previously14,15. The topographies after imprinting were determined and visualized by AFM. As shown in Figure 2A, based on the preparation conditions as depicted in Table 1, wrinkle-like topographies were fabricated with different wavelengths (W; μm) and amplitudes (A; μm). For the wrinkle substrate, the anisotropic wave-like structure could be clearly observed. The amplitude increased with increasing wavelength both these features are coupled and associated with the oxidation degree of the surface, i.e., the time of plasma oxidation treatment.
113 The amplitudes of the topography were 0.05 μm, 0.7 μm, and 3.5 μm for W0.5, W3, and W10, respectively. The different substrates with the aligned topographies are denoted as W0.5, W3, and W10. Flat was used as a control.
After the fibroblast culture and subsequent decellularization, the remaining CDM had a significant influence on the surface topography of the substrate (Figure 2B). For Flat, compared to the smooth surface before CDM deposition (original), the surface with CDM showed a much rougher surface structure indicating the presence of a newly added layer. For W0.5, intriguingly, the CDM completely covered the original wave-like structure, which could no longer be observed. For W3, the topography was still clearly distinguishable after CDM deposition although the amplitude decreased from 0.7 μm to about 0.4 μm indicating that more CDM was collected at the bottom of the wave-like structure. The change in roughness was not clear on the W10 substrate, which may be due to the larger dimension but also here the amplitude decrease substantially going from 3.5 μm to about 2.2 μm.
Figure 2. Representative AFM images of substrate and topography profiles (height) of the structured PDMS substrates obtained (A) after imprinting and (B) after ECM deposition by fibroblasts with subsequent decellularization. W0.5, W3, and W10 stands for W0.5/A0.05, W3/A0.7, and W10/A3.5, respectively, and W is the abbreviation of wavelength.
To further confirm that the visualized layer on top of the substrates using AFM was indeed the decellularized ECM, two major ECM glycoproteins (Fn, Col I) were stained by immunofluorescence. Both proteins were found to be present in the CDM, suggesting the maintenance of bioactivity in the fibroblast-derived ECM. As illustrated in Figure 3, the ECM proteins displayed anisotropic structure (along the direction of wrinkle) on all the substrates except Flat, which showed isotropic fiber structures. With increasing wrinkle size, the orientation degree of Fn (Figure 3A) and Col I (Figure 3B) increased. Furthermore, the ECM proteins were organized into a network, indicating that CDM organization and structure was well retained after decellularization.
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Figure 3. Representative immunofluorescent image of macromolecular ECM components (A: Fn; B: Col I) after decellularization. The white color arrows refer to the direction of wrinkle. Scale bar is 40 μm. Corresponding angular graph of Col I orientation on different substrates (C), statistical analysis of Col I orientation (D), and quantified fluorescence intensity of Col I compared to the mean values of Flat substrate (E). Five images for each substrate were analyzed. Data are shown as mean ± standard deviation (SD), and N.S, not significant, **P < 0.01, ***P < 0.001.
To further confirm the discrepancy between different substrates, the orientation distribution of Col I fiber was measured (Figure 3C). Compared with the broad orientation distribution of Col I for Flat and W0.5, there was a narrow distribution for W3 and W10, indicating the higher level of orientation. We also quantitatively analyzed the orientation of Col I, calculated as the percentage of the main axis of fiber within 10°from the direction of topography44,45. As shown in Figure 3D, W3 and W10 displayed the highest degree of orientation (65%, 61%, respectively), much higher than that for W0.5 (42%). The Flat control displayed no specific orientation, which is to be expected as there is no surface structuring present. Furthermore, the fluorescent intensity for Col I (Figure 3E) and Fn (Figure S3), major components in ECM, were quantified by Fiji. The results showed that no significant difference were found for the various samples, suggesting that the amount of deposited CDM was similar on all substrates. These results demonstrate that the CDM maintains the proper morphology after decellularization, and that substrate topography has a noteworthy influence on the orientation of deposited Fn and Col I in CDM. The orientation of the CDM most likely
115 results from the initial orientation of the cultured fibroblast that also responded well to the W3 and W10 anisotropic structures (Figure S1).
7.3.2 hBM-MSCs morphology on CDM-deposited topography substrates
Morphology and structure, such as cell area and cell orientation, are important factors in the function of native tissues and organs on both biological and mechanical levels46. For identifying the influence of deposited CDM on morphology of the cells, hBM-MSCs were cultured on pristine topography and the substrates coated with CDM after decellularization, and allowed to adhere and attach/spread for 1 day. To analyze cell nucleus and cytoskeleton, cells were stained with DAPI and phalloidin, respectively. Cell orientation, calculated as the percentage of the cells that have their main axis within 10°of the topography direction 44,45, was determined using Fiji.
Illustrated in Figure 4A and 4B, the orientation of hBM-MSCs was highly influenced by the topography and CDM. Fibroblasts grown on the different substrates were also stained after culturing for 1 day and 10 days, which is shown in Figure S1. The highly oriented fibroblasts resemble also the high degree CDM alignment. Apparently, deposited CDM follows the orientation of the cells. For the pristine substrates of Flat and W0.5 without a CDM coating, hBM-MSCs were randomly oriented (the orientation degree was 22% (randomly chosen direction as there is no surface topography direction) and 36%, respectively, Figure 4C) after 1 day. In contrast, cells on W3 and W10 displayed higher degree of orientation (69% and 94%, respectively) along the direction of the topography. For the substrates on which the deposited CDM resides, there is a slight increasing in orientation level for W0.5+CDM (51%) than the pristine substrates, indicating that CDM could facilitate the orientation of hBM-MSCs for the smaller wrinkle surface (W0.5). This indicates that even though the CDM layer is covering the W0.5 topographies and are not identifiable anymore, the alignment of the fibroblasts still deposits the CDM in the direction of the wrinkles be it less pronounced that for the W3 and W10. The CDM is then most likely acting as another topography substrate and guiding the cell orientation with the anisotropic protein fibers of the CDM (as shown in Figure 3A, 3B). In contrast, the cell orientation decreased for W3+CDM (53%) compared with W3, possibly due to the decreased amplitude after CDM deposition, which may diminish the response to the topography. However, there is no change in orientation distribution for W10 and W10+CDM.
The average single cell area (area/cell, μm2) was also quantified as it is well known that cell adhesion and spreading is able to influence the expression of differentiation markers of stem cells47. As shown in Figure 4D, for pristine wrinkle substrates, the average area/cell gradually decreased with increasing wrinkle dimensions. The cell area was 1742 μm2, 1659 μm2, 1368 μm2 and 1202 μm2 for Flat, W0.5, W3, and W10, respectively. Interestingly, the area/cell slightly decreased after CDM deposition. For Flat+CDM, W0.5+CDM, W3+CDM, and W10+CDM the cell area was about 1583 μm2, 1480 μm2, 1230 μm2, and 1030 μm2, respectively. The reason for the decreasing cell area for each substrate, for instance, Flat versus Flat+CDM, may be attributable to the CDM deposition, increasing the roughness of substrate where cells grown on and leading to the limitation of cell spreading. Furthermore, the density of hBM-MSCs was quantified for 1 and 14 days after seeding and the results indicate that no significant difference was found for the pristine substrates compared to substrates coated with CDM (Figure S2). Cell aspect ratio (CAR) was also quantified (Figure 4E) and for cells cultured on W0.5, W3, and W10, the CAR was 7.5, 11, and 18.3, respectively, much higher than cells grown on Flat (CAR of 5.3). Intriguingly, substrates with deposited CDM enhanced the cell elongation. CAR for Flat+CDM, W0.5+CDM, and W3+CDM was 9.8, 10.5, and 16, respectively. However, there is not much difference for W10 versus W10+CDM. Collectively, these findings elucidate the significant influence of CDM, the native ECM, on the cell orientation, cell area, cell elongation, and shows that the addition of CDM does not automatically leads to more spreading.
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Figure 4. Representative fluorescence microscopy images of hBM-MSCs grown on (A) pristine topography and (B) substrates deposited CDM for 1 day, respectively. The row below the fluorescent image was the corresponding angular graph of cell cytoskeleton orientation on different substrates. The cytoskeleton (red) and the nucleus (blue) were visualized using TRITC-labeled phalloidin and DAPI as staining, respectively. The arrow represents the direction of the wrinkle. Scale bar is 100 μm for all the images. Statistical analysis of (C) cell orientation, (D) area per cell, and (E) cell aspect ratio (n ≥ 60 cells, three independent experiments). Data are shown as mean ± standard deviation (SD), and *P < 0.05.
7.3.3 Topography and CDM display synergistic effect on MSC osteogenesis
To determine the synergistic effect of topography and CDM on osteogenesis of hBM-MSCs, these cells were cultured on the original wrinkle substrates and substrates bearing CDM, respectively, and using osteogenic induction medium (OM) as culture medium for 14 days. After that, cells were fluorescently labeled for osteopontin (OPN), a well-documented marker expressed in the later process of osteogenesis48. The cells stained by OPN were imaged by TissueFaxs, which is a high-throughput imaging method that provides the possibility of maintaining the same parameters during the whole imaging process allowing for the fluorescence output to be compared appropriately for quantification. As shown in Figure 5A, cells grown on Flat and W10 compared to those on W0.5 and W3 exhibited a higher expression level of OPN. Interestingly, for cells cultured on the substrates decorated with CDM, the differentiation capacity was
117 enhanced, especially for W0.5 and W3. In contrast, there was no prominent difference for Flat and W10 between pristine substrates and coated with CDM. The effect was further quantified and depicted in Figure 5B, the OPN level of cells on W0.5+CDM and W3+CDM was a 2.72-fold and 1.73-fold increase with respect to those on W0.5 and W3, respectively. In contrast, no significant difference was found between the group of Flat and Flat+CDM, W10 and W10+CDM. These findings suggest that CDM layer could significantly improve the differentiation towards the osteogenic lineage but not in a similar degree for all substrates and that only a specific topography is able to enhance the CDM effect and that CDM by itself only displays a minor contribution without the presence of topography as indicated by the comparison between Flat and Flat+CDM.
Figure 5. Immunofluorescent labeling of osteogenic marker OPN of hBM-MSCs cultured on the original substrate and CDM substrates cultured for 14 days in OM (A). hBM-MSCs were labeled for nuclei (DAPI, blue), and OPN (red). The scale bar is 100 μm for all images. Quantification of OPN expression (B) in cells cultured in OM at day 14, normalized by cell number (n ≥ 100 cells, three independent experiments). Data are shown as mean ± standard deviation (SD), and **P < 0.01, ***P < 0.001.
In order to further confirm the differentiation behavior, the cultures were treated with Alizarin red after a culture of 21 days, which is able to visualize calcium deposition, an important indicator to determine the final stage of osteogenic differentiation49. As shown in Figure 6A, the samples of Flat, W0.5 and W3 were positive for Alizarin red (red color), however, none of the cells cultured on W10 displayed mineral
118
deposition. Interestingly, hBM-MSCs cultured on the substrate coated with CDM showed more mineralized calcium nodules than pristine wrinkle surfaces. To quantify the mineralization of hBM-MSC, calcium deposits were de-stained, and to determine the amount of extracted stain the optical density (OD) was measured at 540 nm. As shown in Figure 6B, W3+CDM displayed the highest OD540, which points towards enhanced osteogenic differentiation capabilities. These results demonstrate that the CDM layer tremendously facilitates the mineralization secreted by hBM-MSCs, but only for distinct substrates and the added positive effects of the CDM differed much among the different substrates. The variations in and amount of the additive effects of the CDM indicates a synergistic effect for the W0.5 and even more so for W3 as the increase is much higher than expected when looking at the contribution of CDM on the Flat substrates and pristine W0.5 or W3.
Figure 6. (A) Representative photographs of Alizarin Red stained calcium nodules indicating extracellular calcium deposits by osteoblasts derived from hBM-MSC cultured for 21 days in OM. (B) Mineralization quantification by elution of Alizarin Red S from stained mineral bone matrix. Data were shown as mean ± standard deviation (SD), and **P < 0.01. Scale bar represents 5 mm.
7.3.4 Enhanced osteogenesis of hBM-MSCs on topography decorated with CDM mediated by mechanotransduction
It is well documented that the fate of stem cells is regulated by physicochemical stimulation from the surrounding ECM via a process of mechanotransduction50, which transduces the physicochemical input into (bio)chemical signals51. To obtain insights about potential signaling proteins involved in transduction of the physiochemical stimuli, we investigated the localization expression of vinculin, Myosin, and YAP
119 both of which have been demonstrated to enhance osteogenic differentiation52–54. From the results of OPN expression and mineral production, the Flat, Flat+CDM, W3, and W3+CDM were selected for further investigation.
The Hippo transcriptional co-activator Yes-associated protein (YAP) has recently been identified as a mechanical rheostat of the cell55, was shown to mediate osteogenic differentiation54,56. Phosphorylation induces the inactivation of YAP in the cytoplasm. Alternatively, activated YAP is translocated into the nucleus, inducing the expression of genes involved in osteogenic differentiation. To investigate topography-induced YAP activation with and without CDM, we immunostained for YAP and quantified the fraction localized to the nucleus of the hBM-MSCs. When cells grown on the substrates coated with CDM, YAP showed a higher enrichment into the nucleus (Figure 7A). Quantitative analysis (Figure 7B) indicated that the cell percentage with YAP located in the nucleus increased from 54 % to 68 % for Flat versus Flat+CDM, and from 60 % to 79 % for W3 versus W3+CDM, respectively. More interestingly, there is no significant difference between Flat and W3. However, for W3+CDM the percentage of nuclear positive cells for YAP is significantly higher than Flat+CDM, indicating that there is a synergistic influence of topography and CDM on YAP localization. These findings indicate that CDM facilitates the YAP translocated from cytoplasm into nuclear and that topography combined with CDM enhance each other.
Figure 7. (A) Representative images of hBM-MSCs residing on different surfaces and the location of YAP after 24 hours of seeding. Blue: nucleus, Green: F-actin, Red: YAP. The arrows refer to the YAP location. Scale bar depicts 20 μm. (B) The percentage of cells with YAP localized in the nucleus. Data are indicated as mean ± standard deviation (SD) (n ≥ 30 cells, three independent experiments), and *P < 0.01.
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Considering the difference for YAP phosphorylation, the related mechanism was investigated on how the sensing of the cell of CDM+topography was related with the YAP localization in hBM-MSCs. Previously, the RhoA/ROCK/myosin-II, the major signaling pathway mediating the contractility of the cytoskeletal in non-muscle mammalian cells was shown to be important for regulating osteogenesis57. Intracellular tension can be characterized by phosphorylated myosin light chain (pMLC). Immunofluorescent staining of pMLC was performed for MSCs after a 1 day culture. Representative immunofluorescent images of hBM-MSCs labeled with pMLC are shown in Figure 8A. For cells grown on W3+CDM it was found that these cells have higher intensity of pMLC in comparison to the other three groups. Quantification of the results indicates that the pMLC level of cells on W3+CDM were 2.1, 1.46 and 1.9-fold higher than on the Flat, Flat+CDM, and W3, respectively (Figure 8B). It was found that there is no difference among the latter three substrates. These results indicate that the CDM layer has a great effect on cell tension or contractility but only when it is combined with the correct topography.
Figure 8. Fluorescent images of cell tension on the various substrates. (A) Representative images of single stem cells on the various substrates with and without CDM in the growth medium cultured for 24 h. Nucleus (Blue), F-actin (Green), pMLC (Red). Scale bar indicates 20 μm. (B) Integrated intensity the fluorescence of pMLC, and compared via normalization for the Flat substrate. Data are given as mean ± standard deviation (SD) (n ≥ 30 cells, three independent experiments), and *P < 0.05.
Vinculin is a marker protein for focal adhesion and interacts with F-actin to recruit actin filaments toward the sites of the focal adhesion.58 Previously, it was shown that focal adhesions (FA) formation is related to RhoA/ROCK signaling pathway by affecting contractility of the cell. Also it was found that more FAs are beneficial for osteogenesis59,60. To investigate the changes for vinculin expression of hBM-MSCs cultured
121 on pristine topography versus CDM+topography, vinculin was stained and visualized by CLSM after culturing for 1 day. As shown in the immunofluorescent image (Figure 9A), vinculin spots with more well-defined dash like structure were identified when cells are grown on Flat+CDM and W3+CDM, compared with Flat and W3, respectively, indicating that CDM is able to enhance vinculin expression. In comparison to the other three substrates, hBM-MSCs on W3+CDM showed the highest expression. In general, micrometer-sized punctate structures are typically regarded as the mature FAs61. To gain more insight in the FAs formation on various substrates, FAs area per cell was quantified using an online Focal Adhesion Analysis Server42 (Figure 9B). The FA area/cell progressively increased on CDM substrates compared to the pristine topography substrates. FA area per cell for cells cultured on W3+CDM (310 μm2) was much larger than those on Flat+CDM (202 μm2), W3 (263 μm2), and Flat (169 μm2). As FAs elongation is an indicator for the maturity62, the elongation was also quantified with the method mentioned above. It was observed that substrates deposited with CDM increased FAs elongation. As illustrated in Figure 9C, FAs elongation for cells grown on Flat+CDM was 2.39, higher than that on Flat substrate (1.87), and there is similar trend for W3 and W3+CDM, varying from 2.3 to 2.75, suggesting that CDM improves the maturity of FAs. Collectively, our findings suggest that CDM on the wrinkle structure is able to facilitate the formation, and elongation of FAs, strengthen the cell contractility and activate more YAP translocated into nucleus and leading to enhanced osteogenesis.
Figure 9. (A) Immunofluorescent staining of hBM-MSCs for vinculin after 1 day culture on various substrates. Blue: nucleus, Green: F-actin, Red: vinculin. Grayscale image for vinculin was shown in Figure S4. Scale bar indicates 20 μm. Quantification of (B) FA area per cell, and (C) FA elongation. The white
122
arrows refer to vinculin spots that are well-defined dash like in structure. Data are displayed as mean ± standard deviation (SD) (n ≥ 30 cells, three independent experiments), and *P < 0.05.
7.4 Discussion
Although, it is well established that both CDM and topography are used to create a microenvironment that mimics the natural niche of stem cells and have a high impact on cellular behaviors. There are relatively little studies that focus on the co-effect of them on osteogenesis of hBM-MSCs, and as far as we could identify, the only one research performed by Zhao et al. elucidate that ECM sheet significantly increase calcium deposition of MSCs, however, the alignment of the these do not seem to be of influence. In our study, the PDMS-based anisotropic topography substrates with varying in wavelength and amplitude were prepared for studying the synergistic effect of CDM and topography on osteogenesis. Furthermore, we found that the enhanced osteogenesis of hBM-MSCs on CDM-topography is partly mediated by focal adhesion, cytoskeletal contractility, and YAP signaling activation.
The topographies used in this study relate to the natural ECM in the sense that it has a fiber-like morphology rather than the shape-edged gratings that often are used. Furthermore, the W0.5 topography represents the nanoscale features found in ECM in vivo, while W3, W10 stand for the microscale features. Previously, these patterns were found to influence hBM-MSC with respect to osteogenic differentiation14. Therefore, in this study, we choose these patterns to further elucidate the synergism between CDM and topography on osteogenic differentiation. For the preparation of extracellular matrix, some previous studies used the ECM scaffolds derived from tissues such as muscle63 and cartilage64. Compared with tissues, cultured cells have several advantages, for example, possibility of mixing ECM harvested from different types of cells, the potential of originating from autologous cells to provide autologous ECM scaffolds to avoid the undesired host responses36. After decellularization, the fibroblast-derived ECM maintain good morphology and network structure (Figure 3A, 3B), and the orientation distribution of ECM fiber is dependent on the dimension of wrinkle (Figure 3C and 3D) indicating strong correlation with the fibroblast response to the wrinkle topography. Although, the main type of the matrix components remained the same, growth factor secretion (e.g., TGF-β1, bFGF, and VEGF) by cells was not considered here but could have an altered composition and be confined within the matrix even after the extensive washing steps. Furthermore, we also demonstrate that there is an important influence of CDM on the cell orientation, cell area and aspect ratio (Figure 4).
Our results are in agreeing well with results from previous studies63, in which Cho et al. demonstrate that topographical and derived ECM have a synergistic effect on myogenic differentiation and maturation. Furthermore, we found that there is a varied degree of OPN expression and mineralization on different wrinkle size substrates decorated with CDM, which is inconsistent with the findings of Zhao and co-workers34. In their study, they cultured stem cells on Flat and nano-patterned substrates of 130 nm in depth and 350 nm in width, deposited with fibroblast-derived ECM, and found that the aligned topography did not influence the osteogenic activity. However, they did not vary the parameter of the topography, therefore overseeing certain possible positive correlations as we present here. With the deposition of the CDM, the fibers also align to some degree to the topography due to the alignment of the fibroblasts that deposited it. From the various results, it can be concluded that the synergy is not due to simply introducing the biochemical nature of the CDM or the alignment of it. Comparing Flat with and without CDM, there is no enhancement in osteogenesis. Therefore, only adding the biochemical nature is not providing substantial stimulus. When aligning the CDM as seen on the W10 substrates, also there alignment with the biochemical nature of the CDM does not enhance differentiation. Only for W0.5 and W3 with CDM, a synergistic effect is observed despite the difference in CDM alignment between the two substrates. While for OPN expression, both these topographies with CDM display a synergistic effect, when looking at the final functional state namely mineralization, the enhancement is more prominent for W3+CDM. That the difference for W0.5 is less efficient as for W3 might be due to the addition of the CDM and thereby masking to some degree the topographical stimulation of the cell, which is still clearly visible for the W3+CDM (Figure 2). We recently showed that topography both the amplitude and wavelength play an important role65 but overcrowding the topography too much will certainly make the stimulus less pronounced. Until now, although some researchers have elucidated that for proliferation, migration, and differentiation on topography66 and stiffness67 of the substrate, YAP-dependent mechanotransduction is required. Few studies investigated the mechanotransduction of cells cultured on CDM. Park et al.28 identified the mechanotransduction of human pluripotent stem cells (hPSC) cultured on fibroblast-derived matrices
123 (FDM) with decellularization, to elucidate cell adhesion, proliferation, migration, and pluripotency. Their results indicate that stiffness of FDM is a dominant influence in mediating hPSC plasticity. Recently, Yang and co-workers68 found that increasing the density of ECM ligands (Fn, Col I, Col IV and laminin) alone can trigger nuclear translocation of YAP without changing substrate stiffness, and further showed that altering the type of ECM modulates hMSC osteogenic differentiation without altering the stiffness of the substrate. Therefore, their findings highlight the important role of ECM in modulating mechanotransduction and differentiation of stem cells. Furthermore, Besenbacher et al.69 found that the adsorption of Fn (a major component of ECM) facilitates focal adhesion formation as compared to the uncoated surface, which is consistent with our current findings (Figure 9). In our study, we not only shown that the combination of CDM and topography synergistically enhances osteogenic differentiation (Figure 5, Figure 6), but also investigated the related proteins involved in the process of mechanotransduction. We demonstrated that for the substrate W3+CDM, the higher expression of OPN and mineralization might be because of the increased formation, and elongation of FAs (Figure 9), stronger cytoskeleton contractility (Figure 8), and more YAP translocated into the nucleus (Figure 7), leading to the expression of the related gene in osteogenic differentiation. The potential mechanism for the improved capacity of osteogenesis on W3+CDM mediated by focal adhesion, cytoskeletal tension and YAP signaling pathway is illustrated in Figure 10. As there are other pathways involved in the osteogenesis process, for example, mitogen-activated protein kinase (MAPK) pathway70, focal adhesion kinase/MAPK and integrin linked kinase/β-Catenin pathways71. Therefore, further investigations are needed to fully identify the mechanisms involved in the osteogenic differentiation process stimulated by CDM and topography.
Figure 10. Schematic representation the effects of topography and Flat substrates coated with CDM to directing osteogenic differentiation. Compared with pristine W3 substrate, wrinkle substrate coated with CDM will enhance the formation and elongation of focal adhesion, strengthen cell contractility, resulting in the activation of YAP and translocation into the nucleus, therefore improving osteogenesis of hBM-MSCs.
7.5 Conclusion
In this study, we prepared anisotropic wrinkle substrate with different wavelength and amplitude, and harvested the cell-derived extracellular matrix to investigate the synergistic effect on the morphology and osteogenesis of hBM-MSCs. We demonstrate that substrates decorated with CDM have a significant impact on cell area and orientation distribution. Moreover, compared to Flat, Flat+CDM, W3, W3+CDM significantly facilitate the fate of hBM-MSCs toward the osteogenic lineage. And this process is connected
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to a higher percentage of cells with YAP localized within the nucleus, stronger cell tension and more formation of focal adhesions. Taken together, this study displays the importance of the ECM in cellular fate decisions and the CDM is able to provide useful approaches to study the interaction between the natural matrix and stem cells, which could facilitate viable applications in tissue engineering and regenerative medicine.
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